Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors.

Hepatoblasts give rise to both mature hepatocytes and cholangiocytes. While Notch signaling has been implicated in the formation of bile ducts composed of cholangiocytes, little is known about the mechanism of lineage commitment of hepatoblasts. Here we describe the role of the Notch pathway in hepatoblast differentiation. Immunohistochemical analysis showed that Jagged1 was expressed in the cells surrounding the portal veins and Notch2 was expressed in most hepatic cells at mid gestation when ductal plates are formed surrounding the portal veins. Interestingly, the Jagged1+ cells were adjacent to ductal plates, suggesting that the Notch signaling is activated in hepatoblasts that undergo differentiation into cholangiocytes. In fact, expression of the Notch intracellular domain in Dlk+ hepatoblasts inhibited hepatic differentiation and significantly reduced the expression of albumin, a marker of both hepatoblasts and hepatocytes. Furthermore, the addition of Matrigel to the hepatoblast culture upregulated the expression of cytokeratin 7 and 19, integrin beta4, and HNF1beta, which are known to be expressed in cholangiocytes. By contrast, downregulation of the Notch signaling by siRNA specific for Notch2 mRNA as well as by the gamma-secretase inhibitor L-685,458 promoted the hepatic differentiation. Consistent with the previous finding that mature cholangiocytes strongly express HNF1beta, but barely express HNF1alpha, HNF4, and C/EBPalpha, activation of the Notch signaling upregulated HNF1beta expression, whereas it downregulated the expression of HNF1alpha, HNF4, and C/EBPalpha. These results suggest that the Notch signaling contributes to form a network of these transcription factors suitable for cholangiocyte differentiation.